The research described in this proposal has two major objectives: (1) to elucidate the special structural and certain of the biological features of the viral and extrachromosomal DNAs, especially those which are covalently closed circular in nature; and (2) to investigate the interaction between DNA and certain DNA-binding enzymes, in particular those which catalyze the rotation of the DNA duplex in response to torsional stress. In pursuit of the first objective we will follow up our discovery that closed circular plasmid DNAs can be generated from certain drug resistance factors in E. coli. We will fully characterize these molecules, both physically and by means of heteroduplex mapping with the original R-factor DNA. We will, in addition, use these DNAs as our primary source of material for further physical work on closed DNA structure. These experiments will include the behavior of closed DNA in denaturing buoyant solvents, including a new method of determining the extent of supercoiling; the kinetics of intercalation of mutagenic drugs into circular DNA; and the mechanism of binding of certain metal-containing drugs, which appear to bind covalently to closed DNA in preference to linear or nicked circular DNA. In furtherance of the second objective we will characterize the DNA nicking-closing enzyme activity which we have discovered in the vaccinia virion. We will analyze the physical properties of the enzyme and attempt to determine the mechanism of its interaction with DNA. We will further investigate the expression of this activity in the vaccina life cycle and attempt to determine the biological function of the enzyme. Finally, we will purify the nicking-closing activity whose presence we have demonstrated in rat liver mitochondria. We will attempt to isolate the nuclear enzyme rat liver from the cells and, by comparison, to establish whether or not the mitochondrial activity results from a mitochondria-specific protein.